Mouse CRISPR Activation Pooled Library (Caprano P65-HSF)
(Pooled Library #1000000113, #92383, #92384)
This mouse CRISPR activation library (Caprano P65-HSF) is in backbone pXPR_502 (P65-HSF). This Caprano library activates over 22,000 mouse genes and is used for genome-wide activation screening.
Each gene activated by this library is targeted by 3-6 gRNAs. These gRNAs are split among the two pools: Set A (Cat# 92383) contains gRNAs 1-3, Set B (Cat# 92384) contains gRNAs 4-6. Each pool has a unique set of gRNAs, but both pools are designed to target the same genes. The slight discrepancy in number of genes targeted between Set A and Set B is because not all genes had 6 gRNAs.
Depositor comments: The modified sgRNA tracr has two MS2 loops and two PP7 loops, although only the PP7 loops are used in this library. This library uses two vectors instead of 3, as the PP7-P65-HSF is on same vector as sgRNA itself. It was found that MS2-P65-HST (hygroR) vector gave poor titer. The 6 guides per gene were designed first based on location relative to TSS, then sequence preferences.
Primers for the lentiGuide backbone can be used with this library in the Sequencing Protocol.
pXPR_502, which uses P65-HSF to activate a gRNA-scaffold.
Genes targeted22,774 (Set A), 22,658 (Set B)
gRNAs67,187 (Set A), 66,889 (Set B)
Controls500 unique non-targeting controls are in each of the two half-libraries (Set A and Set B)
These libraries are delivered as suspended DNA in microcentrifuge tubes on blue ice. A tube's contents will not necessarily be frozen. For best results, minimize freeze-thaw cycles.
Pooled libraries #92383 and #92384 will be delivered as a single tube containing a pooled half-library with 3 gRNAs per gene.
Pooled library #1000000113 will be delivered as two tubes, each containing a pooled half-library with 3 gRNAs per gene (for a total of up to 6 unique gRNAs per gene).
Please visit https://www.biorxiv.org/content/early/2018/06/27/356626 for bioRxiv preprint.
These pooled libraries were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the pooled libraries were described, and include Addgene in the Materials and Methods of your future publications.
Example for your Materials & Methods section:Mouse Caprano CRISPR activation pooled library set A was a gift from David Root and John Doench (Addgene #92383).
Mouse Caprano CRISPR activation pooled library set B was a gift from David Root and John Doench (Addgene #92384).
For your References section:Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Sanson KR, Hanna RE, Hegde M, Donovan KF, Strand C, Sullender ME, Vaimberg EW, Goodale A, Root DE, Piccioni F, Doench JG. Nat Commun. 2018 Dec 21;9(1):5416. doi: 10.1038/s41467-018-07901-8. PubMed 30575746